Escherichia coli is a widely used organism for the expression of heterologous proteins. It easily grows to a high cell density on inexpensive substrates to provide excellent volumetric and economic productivities. Well established genetic techniques and various expression vectors further justify the use of Escherichia coli as a production host. However, a high rate of protein synthesis is necessary, but by no means sufficient, for the efficient production of active biomolecules. In order to be biologically active, the polypeptide chain has to fold into the correct native three-dimensional structure, including the appropriate formation of disulfide bonds.
In many cases, the recombinant polypeptides have been found to be sequestered within large refractile aggregates known as inclusion bodies. Active proteins can be recovered from inclusion bodies through a cycle of denaturant-induced solubilization of the aggregates followed by removal of the denaturant under conditions that favor refolding. But although the formation of inclusion bodies can sometimes ease the purification of expressed proteins; in most occasions, refolding of the aggregated proteins remains a challenge.
Various attempts have been made to improve the folding of heterologous proteins in the bacterial cytoplasm. In addition to the traditional methods, including lowering the temperature of the culture, increasing knowledge of the mechanism and effectors of protein folding has enabled new approaches to solve the problem of aggregation.
Studies in vitro have demonstrated that, for the vast majority of polypeptides, folding is a spontaneous process directed by the amino acid sequence and the solvent conditions. Yet, even though the native state is thermodynamically favored, the time-scale for folding can vary from milliseconds to days. Kinetic barriers are introduced, for example, by the need for alignment of subunits and sub-domains. And particularly with eukaryotic proteins, covalent reactions must take place for the correctly folded protein to form. The latter types of reaction include disulfide bond formation, cis/trans isomerization of the polypeptide chain around proline peptide bonds, preprotein processing and the ligation of prosthetic groups. These kinetic limitations result in the accumulation of partially folded intermediates that contain exposed hydrophobic ‘sticky’ surfaces which promote self-association and formation of aggregates.
Expression of mammalian proteins is more complicated than bacterial proteins because many of them require intramolecular disulfide bonds for their activity. Thus additional effectors such as foldases and proper redox potential are required to achieve their native structures. Even though the periplasmic space of Escherichia coli provides an oxidizing environment as well as folding proteins such as DsbA, B, C, and D, in many cases, simple secretion of complex proteins into the periplasmic space is not sufficient to form correct disulfide bonds.
Accessory proteins known as foldases and chaperones have been found to assist in the proper folding of proteins in vivo. Foldases have a catalytic activity that serves to accelerate rate-limiting covalent steps in folding. Chaperones, on the other hand, perform many functions, the most important of which is to provide an environment for nascent proteins to fold without the competing process of self-association. In addition to the well-characterized molecular chaperones, such as GroEL and DnaK proteins, a number of additional cytoplasmic proteins have been identified to affect the folding of heterologous proteins.
Following the discovery of numerous bacterial or eukaryotic foldases and their specific roles in the oxidation and isomerization of disulfide bonds, many attempts have been made to use those proteins in the periplasmic space or even in the cytoplasm of Escherichia coli (see, for example, Bessette et al. (1999)). The co-expression of molecular chaperones has been shown to partially solve the problem of inclusion body formation in the expression of certain recombinant proteins (see, for example, Richardson et al. (1998) Trends Biochem. Sci. 23:138-143; and Bukau et al. (1998) Cell 92:351-366).
However, the effect of molecular chaperones can be product-specific and the co-expression of each molecular chaperone with the target proteins is often cumbersome. Moreover, in some cases, the expression of a molecular chaperone is detrimental to cell growth. Despite the recent advances, the expression of properly folded mammalian proteins in Escherichia coli still remains as a great challenge. This is mainly due to the difficulties in the control of the key parameters for disulfide bond formation including the sulfhydryl redox potential inside the cells.
For several decades, in vitro protein synthesis, also called cell-free protein synthesis (CFPS), has served as an effective tool for lab-scale expression of cloned or synthesized genetic materials. In recent years, in vitro protein synthesis has been considered as an alternative to conventional recombinant DNA technology, because of disadvantages associated with cellular expression. In vivo, proteins can be degraded or modified by several enzymes synthesized with the growth of the cell, and, after synthesis, may be modified by post-translational processing, such as glycosylation, deamidation or oxidation. In addition, many products inhibit metabolic processes and their synthesis must compete with other cellular processes required to reproduce the cell and to protect its genetic information.
Cell-free protein synthesis has the potential to replace bacterial fermentation as the technology of choice for the production of many recombinant proteins. The most significant advantage is that all of the resources in the reaction theoretically can be directed toward production of the desired product and not to secondary reactions, e.g., those that maintain the viability of the host cell. In addition, removing the need to maintain host cell viability allows the production of proteins that are toxic to the host cell. Furthermore, the lack of a cellular membrane allows direct access to the reaction volume, allowing for addition of reagents that increase the efficacy of the in vitro synthesis reaction (e.g., increase protein yield).
To compete with standard fermentation processes, it is desirable that in vitro synthesis reactions produce equivalent quantities of biologically active proteins at the same (or better) cost (see Voloshin and Swartz (2005) Biotechnol Bioeng 91:516-21). One element of achieving a cell-free synthesis system that competes with fermentation processes is to employ a low cost energy supply for the reaction. To this end, it was found that glucose, the preferred low-cost substrate for bacterial fermentation, could be used in in vitro synthesis if the pH of the system was stabilized (Calhoun and Swartz (2005) Biotechnol Bioeng 90:606-13).
Many industrially relevant proteins, including mammalian proteins, require disulfide bonds for activity. To promote disulfide bond formation, a buffer of reduced (GSH) and oxidized (GSSG) glutathione can be added to an in vitro synthesis reaction to create an oxidizing environment in which disulfide bonds will form. Unfortunately, GSSG is rapidly reduced during in vitro synthesis reactions by two enzymatic pathways mediated by glutathione reductase (Gor) and thioredoxin reductase (TrxB). Deletion of either glutathione reductase or thioredoxin reductase from the strain used to make the extract had little effect on the rate of reduction of GSSG (Kim and Swartz (2004) Biotechnol Bioeng 85:122-9). Deletion of both gor and trxB results in the mutational conversion of the enzyme AhpC from a peroxiredoxin to a disulfide reductase, a mutation which promotes more rapid growth but also stimulates disulfide bond reduction.
To overcome the shortcomings of the gene-deletion systems, iodoacetamide (IAM) has been added to the extract to derivatize the active site cysteines of TrxB and Gor, thereby inactivating those enzymes (Kim and Swartz (2004) Biotechnol Bioeng 85:122-9; U.S. Pat. No. 6,548,276 and U.S. Pat. No. 7,041,479). While IAM-mediated inactivation of TrxB and Gor is useful for promoting disulfide bond formation, conventional IAM treatment can also result in a reduction in protein yields (Kim and Swartz (2004) Biotechnol Bioeng 85:122-9).
Improvements in in vitro synthesis systems that produce active disulfide bond-containing proteins are of continued interest and are the subject of the present invention.